The present invention relates to a process for preparing polynucleotides on a solid support. The present invention also relates to a reactor containing a solid support and to a device including this reactor, which are useful in the process for preparing polynucleotides according to the invention.
The synthesis of polynucleotides on a solid support is particularly used in automated syntheses of DNA or RNA oligonucleotides. In the present application, xe2x80x9cpolynucleotidesxe2x80x9d is understood to mean deoxyribonucleic acid or ribonucleic acid fragments or, more generally, polynucleotides or oligonucleotides where the bases, inter-nucleotide phosphate linkages, or alternatively the ribose rings of the bases, can be chemically modified in a known manner. This may be especially oligonucleotides with xcex1 or xcex2 anomers, oligonucleotides with inter-nucleotide linkage of the phosphorothioate or methyl phosphonate type, or alternatively oligothionucleotides.
The principle for the chemical synthesis of nucleic acids on a solid support is nowadays widely described in the specialist literature, and a number of apparatus are available on the market which perform automatically all or part of the synthesis steps. Among the chemical routes described, only the so-called phosphoramidites method (Caruthers et al.: EP 0,035,719 B1) is up until now sufficiently efficient to envisage the production of nucleic acids on an industrial scale.
The preparation of oligonucleotides or polynucleotides is carried out in a reactor containing a solid support and comprises the treatment of the solid support such as an inorganic polymeric support by a series of successive steps, each of the series leading to the addition of a new nucleotide on the support. The series of successive steps, or synthesis cycles, are carried out as many times as is required by the manufacture of an oligonucleotide or a polynucleotide of desired length.
In a process for synthesizing polynucleotides on a solid support, the solid support traditionally consists of glass beads of controlled porosity (CPG) or, more generally, of particles of a functionalized inorganic or organic polymer.
The techniques conventionally used involve the use of eight different reagents as solid supports, consisting of the said functionalized inorganic or organic polymer linked to a nucleoside A, T, C, G or U, depending on whether the sequence to be prepared contains, as first deoxyribo- or ribonucleotide A, T, C, G or U. Manufacturers therefore supply reactors in which one of these nucleosides has previously been attached to the support. Depending on whether the sequence starts with A, T, C, G or U, the appropriate reactor is then chosen. The elongation of this first nucleoside then occurs in the 3xe2x80x2xe2x86x925xe2x80x2, or 5xe2x80x2xe2x86x923xe2x80x2 direction, using coupling reagents.
Numerous supports have already been described in the literature for the solid phase synthesis of oligonucleotides.
There may be mentioned organic polymers such as polystyrene (Nucleic A. Res. 1980, volume 8), polyacrylamide acryloylmorpholide, polydimethylacrylamide polymerized on kieselguhr (Nucleic Ac. Res. 9(7) 1691 (1980)).
Other supports described are of inorganic nature, in particular based on silica functionalized by a hydrocarbon radical carrying an NH2 and/or COOH group (J. Am. Chem., 105, 661 (1983)), or the support based on silica functionalized by a 3-aminopropyltriethoxysilane group whose use in phosphite and phosphoramidite synthesis for the preparation of oligonucleotides was described for the first time in European Patent No. 0,035,719.
There is known, from French Patent Application FR 93 08 498 and PCT/FR94/00842, a process for the solid phase synthesis of oligonucleotides in which a so-called xe2x80x9cuniversalxe2x80x9d support is used, that is to say a solid support which can be used regardless of the first nucleotide of the RNA or DNA to be synthesized, regardless of the type of monomeric reagent used during the synthesis, that is to say regardless of the type of substitution on the phosphate group in 3xe2x80x2 or in 5xe2x80x2, depending on whether the synthesis is carried out in the 5xe2x80x2xe2x86x923xe2x80x2 or 3xe2x80x2xe2x86x925xe2x80x2 direction.
In particular, the xe2x80x9cuniversal naturexe2x80x9d of the solid phase supports can be obtained by functionalization of the inorganic or organic polymer with a hydrocarbon radical containing glycol type groups in which an OH group and a nucleophilic group are present in the vicinal position, that is to say on two adjacent carbons, at the end of the hydrocarbon radical, it being possible for these two carbons to be optionally substituted by inert groups. xe2x80x9cInert groupxe2x80x9d is understood here to mean a group which does not react under the conditions encountered during the various steps of the synthesis on a solid support of nucleic acids according to the invention.
In a specific embodiment, a process for synthesizing polynucleotides comprises the following steps of:
1) condensing the OH group in 5xe2x80x2 or 3xe2x80x2 of the first nucleotide or of an oligonucleotide linked at its other 3xe2x80x2 or 5xe2x80x2 end to the said solid support, by means of a coupling agent, with the phosphate group optionally substituted respectively in position 3xe2x80x2 or 5xe2x80x2 of a monomeric nucleotide reagent protected in 3xe2x80x2 and 5xe2x80x2;
2) oxidizing or sulfurizing the phosphite type internucleotide linkage obtained in step 1) into a phosphate linkage respectively;
3) deprotecting the 5xe2x80x2-O or 3xe2x80x2-O end of the product obtained in step 2);
4) repeating steps 1) to 3) as many times as there are nucleotides to be added in order to synthesize the nucleic acid.
The above steps lead to an oligonucleotides linked to the solid support. The process comprises a final step of detaching the nucleic acid from the support and removing the groups for protecting the bases and, where appropriate, the 2xe2x80x2-O positions of the nucleic acid.
In the techniques where the solid support is already linked to a first nucleoside corresponding to the first nucleotide of the sequence to be prepared, before the start of the synthesis cycles, the said support generally contains a protection in 5xe2x80x2 or 3xe2x80x2 of the said nucleoside. In this case, the synthesis cycle starts with a deprotection step in acidic medium, in general a detritylation.
According to the variants used most commonly, the said monomeric nucleotide reagent corresponds to the formula: 
in which:
A represents H or an optionally protected hydroxyl group,
is a purine or pyrimidine base whose exocyclic amine functional group is optionally protected,
C is a conventional protective group for the 5xe2x80x2-OH functional group,
x=0 or 1 with
a) when x=1:
R3 represents H and R4 represents a negatively charged oxygen atom, or
R3 is an oxygen atom and R4 represents either an oxygen atom or an oxygen atom carrying a protecting group, and
b) when x=0, R3is an oxygen atom carrying a protecting group and R4 is either a hydrogen or a disubstituted amine group,
when x is equal to 1, R3is an oxygen atom and R4 is an oxygen atom, the method is in this case the so-called phosphodiester method; when R4 is an oxygen atom carrying a protecting group, the method is in this case the so-called phosphotriester method,
when x is equal to 1, R3is a hydrogen atom and R4 is a hydrogen atom and R4 is a negatively charged oxygen atom, the method is in this case the so-called H-phosphonate method, and
when x is equal to 0, R3is an oxygen atom carrying a protecting group and R4 is either a halogen, the method is in this case the so-called phosphite method and, when R4 is a leaving group of the disubstituted amine type, the method is in this case the so-called phosphoramidite method.
The steps of a cycle of synthesis by the phosphoramidite method are conventionally the following:
1) condensation of the 5xe2x80x2 terminal hydroxyl of a nucleoside or of an oligonucleotide covalently attached to the solid support with a phosphite type compound according to the reaction: 
2) oxidation of the phosphite bond obtained to a phosphate according to the reaction: 
3) blocking of the hydroxyl groups of the unreacted nucleosides;
4) liberation of the 5xe2x80x2 terminal hydroxyl from the last nucleoside so as to generate an active site for the next synthesis cycle.
Each nucleotide is sequentially added to the support by repeating steps 1 to 4. At the end of the synthesis, the oligonucleotide is separated from the support and freed of its protecting groups by a controlled hydrolysis reaction.
Commercial synthesizers specialized in the synthesis of oligonucleotides are designed so as to automatically carry out the synthesis steps described above. These synthesizers are generally composed of a reactor containing the solid support, a reagent mixer, one or several unit(s) for selecting the reagents and the vessels containing the said reagents. The synthesis steps are carried out by successively adding the selected reagents to the reactor. Most often, the solid support is washed with acetonitrile after each step.
The solid support is not immobilized and does not occupy the whole volume of the reactor but, in general, only half of the volume of the reactor, and in any case no more than three quarters, so as to allow adequate stirring of the solid phase.
The reactor, as used in commercial synthesizers, has the shape of a vessel crossed by the flow of reagents which causes the stirring (or fluidization) of the solid support. It is indeed considered that the stirring of the solid phase is essential because it allows better penetration of the solvents and reagents into the pores of the solid phase generally consisting of porous beads or porous membranes (see Methods in Molecular Biology: Protocols for Oligonucleotides and Analogsxe2x80x94Synthesis and Propertiesxe2x80x94Edited by Sudhir Agrawalxe2x80x941993xe2x80x94Humana Press; Totowa, N.J., pages 442-444 and 454).
The mixer is situated upstream of the reactor, connected to it by a pipeline. The units for selecting the reagents generally consist of electrovalves and allow selective opening of the inlets/outlets of the hydraulic circuit as well as the routes for the passage of the reagents. The cohesion of the hydraulic system is ensured by a series of suitably connected tubes or capillaries. It is recommended to install the equipment in an air-conditioned room at 20xc2x0 C., the temperature at which the reactions are performed.
While the efficiency of the reactions is sufficient under these conditions, the duration of the reactions and the required excess of reagents in order to carry out good washing by successive dilutions are the principal disadvantages of the synthesizers as described above. In this case, the consumptions of reagents and the duration of the syntheses are substantially greater than the values calculated on the basis of known laws of organic synthesis. These disadvantages were attributed to the limiting factor which the rate of diffusion of the reagents in the pores constitutes.
The aim of the invention is the improvement of the efficiencies of existing methods of synthesis of oligonucleotides.
The method according to the present invention consists in a modification of organic synthesis apparatuses and parameters so as to improve the productivity thereof. The improvements affect in particular the duration and the consumption of reagents necessary for carrying out a synthesis.
The subject of the present invention is also a reactor for preparing polynucleotides according to a process of the invention, the reactor being in the form of a column containing a solid support through which the solutions of reagents and/or solvents are circulated, wherein the solid phase constituting the solid support is immobilized in said reactor such that said solutions migrate in the column and through said solid phase according to a frontal progression, the successive solutions of each step of a synthesis cycle not mixing at all or very little.
In particular, the subject of the present invention is therefore a reactor which is useful in a process for preparing polynucleotides on a solid support, which consists of a cylindrical column completely filled with particles of porous materials constituting a solid support as described above.
The subject of the present invention is finally a device for the synthesis of polynucleotides on a solid support containing a thermostatted reactor and a thermostatted collector which performs the collection, the heating to the temperature of the reactor, then the mixing of the reagents before their introduction into the reactor.
Other advantages and characteristics of the present invention will appear in the light of the detailed description below.